Eukaryotic DNA replication; Chromosome structure & maintenance; Heterochromatin organization; Cell cycle control

The initiation of DNA replication in eukaryotic cells is a highly regulated process that leads to the duplication of genetic information for the next cell generation.  DNA replication, which occurs during S phase of the cell cycle, is intimately linked to mitotic progression and eventually cell division.  Inaccurate DNA replication in turns leads to abnormal chromosome segregation resulting in aneuploidy and genomic instability, a hallmark of most cancerous cells.  Thus the accurate duplication of DNA is of paramount importance and is governed by a number of proteins including the Origin Recognition complex (ORC) which serves as a landing pad for the assembly of a multiprotein pre-replicative complex.  Other than its bonafide role in DNA replication, ORC proteins are involved in diverse functions including gene silencing, heterochromatin organization, cytokinesis and also in dendrite formation in postmitotic neurons. The focal point of research in my lab is to study the events and uncover the cues that integrate DNA replication with heterochromatin organization, chromosome segregation and cytokinesis, major focus being on the role of ORC in interconnecting these events.

The research project includes:

Role of ORC proteins in heterochromatin organization and chromosome structure

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ORC proteins have been implicated in heterochromatin organization in mammalian cells (Figure 1) and Drosophila and gene silencing in Saccharomyces cerevisiae. To further pinpoint the role of ORC subunits at heterochromatin, my lab focuses on the identification of the interacting partners of ORCs at heterochromatin during different stages of the cell cycle and to delineate the replication function of ORC proteins versus their role in chromatin compaction.

It has been suggested that centromeric heterochromatin is necessary for both the cohesion of sister chromatids and the subsequent normal disjunction of mitotic chromosomes.  In the fission yeast Schizosaccharomyces pombe, the homologue of the heterochromatin protein (HP1) protein Swi6 is critical for efficient sister chromatid cohesion during cell division.  The loss of Orc2 and Orc3 proteins from human cells also results in the loss of HP1 (HP1a and b) from the centric heterochromatin during mitosis.  In Orc2 depleted cells, a population of cells arrests in mitosis with defective chromosome structure and condensation, spindle and chromosome congression defects.

How the Orc2 depletion results in the abnormal localization of HP1 proteins from mitotic chromosomes remains to be addressed.  Studying the localization of proteins which interact with ORC at heterochromatin with respect to differential levels of ORC proteins as well as dissecting the functional significance of their interaction will be critical towards understanding how ORC is involved in organizing chromatin structure during different stages of the cell-cycle. Further, live cell imaging using mitotic markers will enable to study the order of the mitotic defects in ORC depleted cells.

Also, structure function analysis of ORC and the heterochromatin interacting partners would provide better insights into the understanding of ORCs involvement in heterochromatin organization and chromosome structure.

Role of Orc6 proteins in cell-cycle progression and cytokinesis

The second main area of the research focus of my lab is to define the role of the replication initiator protein Orc6 in cytokinesis and its interaction with proteins that govern the chromosome and cell division cycle in human cells.

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Although Orc6 depletion results in abnormal cytokinesis, how Orc6 functions in coordinating chromosome segregation and cytokinesis is not understood. Proteins interacting with Orc6 during different stages of the cell-cycle would throw light on how Orc6 is involved in cell-cycle progression. Further, the mechanism that results in cytokinesis defects in the absence of Orc6 and the stages at which Orc6 executes its function remains to be elucidated. Time-lapse imaging experiments will be used to investigate the mechanism of Orc6 function. Further, genetic complementation experiments will be used to delineate the replication function and the cytokinesis function of Orc6 in human cells.

ORC and other human replication initiation proteins have been shown to be required for replication of Epstein Barr virus (EBV), a virus that is associated with Burkitt’s lymphoma, immunoblastic B-cell lymphoma and Hodgkin’s diseas. Deletion of ORC genes has been observed in non-Hodgkin’s lymphomas, uterine leiomyomas and malignant myeloid disease. By increasing our understanding of the involvement of ORC in critical cellular processes including cell chromosome duplication and segregation, we will gain valuable insight into how genetic instability contributes to cancer progression.